Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

Microbial conversion of solasodine to 1-androstene-3,17-dione (AD), a key intermediate for androgen synthesis

The spirosolane side chain of solasodine has been cleaved by cholesterol preinduced Mycobacterium sp. NRRL B-3805 to yield 1-androstene-3,17-dione (AD), a key intermediate for the synthesis of androgenic drugs. Conversion up to 34% has been recorded in shake flask culture after 192 h incubation period using dimethyl-formamide as carrier for solasodine addition.     

Microbial Degradation of Sterols

A process is described for the microbial degradation of cholesterol and plant sterols, to produce androsta-1, 4-diene-3, 17-dione and androst-4-ene-3, 17-dione, by two newly isolated bacteria designated Mycobacterium sp. NRRL B-3683 and Mycobacterium sp. NRRL B-3805. These myocbacteria produce substantial amounts of 17-ketonic compounds without appreciable degradation of the steroid nucleus. No ring degradation inhibitory agents are necessary. The first microbiological production of 20alpha-hydroxymethylpregna-1, 4-dien-3-one is also reported.     

Testosterone as biotransformation product in steroid conversion byMycobacterium sp.

Summary Testosterone production byMyc. sp. NRRL B-3683 is discussed. The unexpected finding that testosterone is not formed by single reduction of 17-keto group of 4-androstene-3,17-dione (AD) but by a double reduction of both 17-keto group and 1–2 doble bound of 1,4-androstadiene-3,17-dione (ADD) is presented.     

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.     

分枝杆菌甾酮C1,2位脱氢酶基因敲除的研究

利用分枝杆菌对植物甾醇进行边链降解可产生4-AD（4-烯-雄甾-3,17-二酮）和ADD（1,4-二烯-雄甾-3,17-二酮）,ADD由4-AD在C1,2位脱氢酶(ksdD)作用下脱氢产生,这两种物质在化学结构上高度相似,难以分离。本文首先扩增出部分ksdD基因,大小为631bp,并以此为基础构建打靶载体pUC19-MK。将pUC19-MK电转分枝杆菌感受态,通过同源重组敲除分枝杆菌染色体上正常的ksdD基因,使C1,2位脱氢酶失活,以达到4-AD大量积累的目的。结果通过初筛筛选出5株转化子,进行甾体转化实验,发酵144h时,1号转化子的4-AD生成率达到17.52％,比出发菌株提高了192％,而此时ADD的生成率仅为6.12％,比出发菌株降低了89.9％。     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using immobilized growing cells of Mycobacterium sp. NRRL B-3683 adsorbed on solid carriers

Summary Living cells of Mycobacterium sp. NRRL B-3683 were immobilized by adsorption on different types of solid carriers in order to produce androsta-1,4-diene-3,17-dione (ADD) from cholesterol. Activated alumina proved to be the most preferred carrier for long-term operation when glucose and peptone were added to the reaction medium. In a repeated-batch process, the maximum productivity of ADD was about 0.19 g/l per day with a molar conversion rate of 77% when 1.0 g/l of cholesterol was added to the reaction medium. The half-life of the immobilized cells was more than 45 days and the system could be reactivated by incubating the immobilized cells in a cell growth medium.     

Dependence of steroid 1(2)-dehydrogenation on the C-17 side chain during cholesterol metabolism by immobilized Mycobacterium fortuitum

1(2)-Dehydrogenation of 4-cholestene-3-one by immobilized Mycobacterium fortuitum NRRL B-8153 and free growing Mycobacterium sp. NRRL B-3805 and Micromonospora chlacea MTCC 329 depended on the nature of the C-17 side chain. The 1(2)-dehydrogenation did not appear to occur before C-17-side-chain cleavage of the 4-cholestene-3-one.S. Patil is and A. Srivastava was with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road Campus, Indore-452 001, India. A. Srivastava is now with the Shripati Singhania R&D Centre. J. K. Pharmaceuticals. 13th Mile Stone. Mathura Road, Faridabad (Haryana) 121 003, India.     

Optimization of androstenedione production in an organic–aqueous two-liquid phase system

A systematic evaluation of the effect of key operational parameters on the selective cleavage of sitosterol to 4-androstene-3,17-dione (AD) with Mycobacterium sp. NRRL B-3805 in a dioctyl phthalate: aqueous buffer two-liquid phase system was performed. Of the parameters assessed, buffer composition, biomedium pH, temperature, and biomass and substrate concentration were those that mostly affected overall bioconversion rate. The optimum pH was 7.5 with Tris buffer. The highest bioconversion rate was observed at 35 °C, although at 40 °C bioconversion activity was virtually lost. Michaelis–Menten type kinetics adequately described the bioconversion system. Increasing biomass concentration from 10 to 70 g_{wet cell weight} l⁻¹ favored AD final yield, although the specific AD yield slightly decreased.     

Growth and cholesterol oxidation by Mycobacterium species in Tween 80 medium.

Mycobacterium strain DP was isolated from marine coastal sediment and tested for its ability to oxidize cholesterol in Tween 80-cholesterol (2.59 mM) medium. Strain DP degraded cholesterol to 4-cholesten-3-one (cholestenone), 4-androsten-3,17-dione (AD), 1,4-androstadien-3,17-dione (ADD), testosterone, and 1-dehydrotestosterone (DHT). Cholesterol disappeared in about 4 days. Cholestenone, AD, testosterone, and DHT accumulations were transient with peak concentrations of 300, 600, 30 to 40, and 21 microM. ADD production peaked after 6 days with a concentration of 1,100 microM. Peak ADD concentrations and production rates compared well with those reported for strain NRRL B3683 on cyclodextrin medium. Tween 80 medium was superior to finely dispersed cholesterol particles for both strains. In comparison, NRRL B3683 (patented for its ability to accumulate AD and ADD) on Tween 80 medium transiently accumulated more AD (approximately 1,000 microM) than did strain DP, but ADD accumulations (200 microM) were significantly lower than those for strain DP. Strain DP could be adapted to grow on ADD, which was initially inhibitory at 3.25 mM. ADD-adapted strain DP cultures produced approximately four times as much DHT from ADD than unadapted cultures did from cholesterol, showing that additional manipulation might enhance testosterone production. We believe that ADD toxicity might account for the low ADD accumulations by NRRL B3683 in Tween 80 medium.     

Mycobacterium sp．高效转化植物甾醇为雄甾-1，4-二烯-3，17-二酮的诱变选育

采用紫外线、亚硝基胍复合诱变雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）的转化产生菌Mycobacterium sp．,结合平板筛选,获得一株遗传性状稳定单产ADD的突变菌株Mycobacterium sp．-11,其ADD质量浓度达到1246ms／L,比原始菌株（484mg／L）提高了150％,经初步优化后发酵液中ADD最高达到1430mg／L,发酵液中ADD质量占ADD、AD两产物质量总和的比例由70％提高到99．1％。     

Metabolism of androst-4-en-3,17-dione by the filamentous fungus Neurospora crassa

Microbial transformation of androst-4-en-3,17-dione (AD; I) using Neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (II), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (III), 7alpha-hydroxyandrost-4-en-3,17-dione (IV), 9alpha-hydroxyandrost-4-en-3,17-dione (V), 14alpha-hydroxyandrost-4-en-3,17-dione (VI), and androst-4,6-dien-3,17-dione (VII). The steroid products were assigned by interpretation of their spectral data such as (1)H NMR, (13)C NMR, FTIR, and mass spectroscopy. The characteristic transformations observed were C-6beta, C-7alpha, C-9alpha, C-14alpha hydroxylations, and C6-C7 dehydrogenation. The best fermentation condition was found to be 6-day incubation at 25 degrees C and pH value of 5.0-6.5 according to TLC profiles. Time course study showed the accumulation of V and VI from the third day and IV from the fourth day of the fermentation. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 3.5mM in one batch. Biotransformation was completely inhibited in a concentration above 7.0mM.     

Isolation and identification of a new metabolite of microbial conversion of upgraded neutral fraction of Polish tall oil by means of four strains of Mycobacterium sp.

Summary A new metabolite, 5-alpha-androstane-3,6,17-trione, was isolated as a product of microbial conversion of upgraded neutral fraction of the Polish tall oil byMycobacterium NRRL B-3683, NRRL B-3805, MB 3683, and MB 3805.     

Microbial oxidation of dehydroepiandrosterone and related compounds.

The oxidation of dehydroepiandrosterone (DHEA), 4-androstene-3, 17-dione, and estrone with Streptomyces roseochromogenes NRRL B-1233 was studied. The oxidation products were isolated and identified as as 16alpha-hydroxy-DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 16alpha-hydroxyestrone. The yields of these three products were 85%, 41% and 18%, respectively. This indicates the substrate stereospecificity of 16alpha-hydroxylase of the organism. An interrelationship between cell growth and the formation of 16alpha-hydroxylated steroid was observed in any case. For formation of 16alpha-hydroxy-DHEA, 16alpha-hydroxylase showed good activity at DHEA concentration of 3.47 x 10(-4)M. In the case of DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 5-androstene-3beta, 16alpha, 17beta-triol were obtained after the yield of 16alpha-hydroxy-DHEA reached the maximum yield for about 30 hr. The oxidation pathway of DHEA is discussed.     

Microbial introduction of a 16 alpha-hydroxyl function into the steroid nucleus.

The introduction of a 16 alpha-hydroxyl function into the steroid nucleus was studied in resting cells of Streptomyces roseochromogenes NRRL B-1233. The oxidation product of dehydroepiandrosterone (DHEA) was identified as 16 alpha-hydroxy DHEA by using thin-layer and gas-liquid chromatography. A linear relation between cell concentration and 16 alpha-OH-DHEA formation was observed. 16 alpha-Hydroxylase showed good activity at pH 8.0 for 16 alpha-OH-DHEA formation. The enzyme showed good activity at 3.1 x 10(-4) M DHEA. The oxidation products of pregnenolone, 4-androstene-3,17-dione, estrone, and 5-androstene-3 beta,17 beta-diol as well as of other substrates were identified as the 16 alpha-hydroxy steroid, respectively. The rates of microbial 16 alpha-hydroxylation were as follows: 76.9% for DHEA, 50.4% for pregnenolone, 43.9% for 4-androstene-3,17-dione, 34.3% for estrone, and 19.6% for 5-androstene-3 beta,17 beta-diol. The organism tested catalyzes 16 alpha-hydroxylation of a wide variety of steroids.     

Glucose and lactose effect on AD and ADD bioconversion by Mycobacterium sp.

Summary The glucose and lactose effect on the steroid biotransformation reactions by Mycobacterium sp. NRRL B-3683 was studied. The reduction reactions increased at pH 7 and when sugars are added. The oxidation reactions are highers at pH 8 without any sugar added. The dehydrogenation reaction is inhibited when an endogenous carbon source is present in the media.     

Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in Bis-(2-ethylhexyl) phthalate

The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.     

Isolation of a biodegradable sterol-rich fraction from industrial wastes

Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96-98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation

A novel two-step transformation process for the production of androsta-l by microorganisms-diene-3,17-dione (ADD) from a high concetration of cholesterol by microorganisms is proposed. Cholesterol (20 g/l) was initially converted to cholest-4-en-3-one (cholestenone) by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/l per day and the molar conversion rate was 80%. Subsequently, a fine suspension of cholestenone (50 g/l), which was prepared directly from the fermentation broth of A. simplex, was converted to ADD by Mycobacterium sp. NRRL B-3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/l per day and the molar conversion rate was 35%. Correspondence to: W.-H. Liu